The long-term objective of the proposed research is to understand better the cellular processes in acetylcholine biosynthesis, secretion, and neuronal function. Despite intensive study of the cholinergic nervous system, it is poorly understood at the molecular level. With the complete nucleotide sequence and a functional promoter for the C. elegans acetylcholine biosynthesis and synaptic vesicle transporter genes (cha-1 and unc-17 respectively), we now possess the tools to conduct a powerful molecular and genetic analysis of cholinergic function in nematodes. The organization of these two genes in a eukaryotic operon is quite unique; the unc-17 coding sequence is completely nested within the first intron of cha-1, and their mRNAs are produced by alternative splicing of a common precursor RNA. This overall structure is conserved from nematodes to mammals, indicating that it may be crucial for regulation. The research proposed herein seeks to understand the regulation of cha-1/unc-17 expression, as well as the function of specific subsets of cholinergic neurons in C. elegans behavior.